Vaccinia, the prototype of the poxvirus family, is a large and complex virus. The virion encapsidates the 185 kb genome and sufficient enzymes to initiate the transcription of early viral genes immediately upon infection. Infection is accompanied by the rapid shut-off of host transcription and replication; early transcription is followed by DNA replication, which is an obligate step for the subsequent switch to the transcription of late viral genes. The virus replicates entirely within the cytoplasm of infected cells; this physical autonomy from host nuclear functions is mirrored in the apparent genetic autonomy of the virus. Vaccinia appears to encode all of the functions required for nucleic acid metabolism. The genome is a linear duplex with covalently-closed hairpin termini reminiscent of yeast telomeres. The autonomy of the virus from host functions, the ease of preparation of biochemically pure material, and the recent advances in the molecular biological analysis of the genome make it an excellent model system for the study of DNA replication. We propose a broad analysis of viral replication which combines genetic, molecular and biochemical analyses. Our aims include: (1) The identification and characterization of viral genes encoding known enzymes with a role in DNA metabolism. (2) The molecular genetic analysis of genes affected in known vaccinia replication mutants. (3) The isolation of additional mutants with replication defects, using classical genetic techniques as well as the targeting of mutations to defined DNA fragments. (4) The refinement of a system for the in vitro mutagenesis and expression of the vaccinia DNA polymerase gene. These experiments will facilitate structure-function analysis and ready the technique for application to genes whose function has eluded classical genetic study. (5) Refinement of an in vitro replication system to aid in the study of replication mutants and the identification of replication functions. These studies should provide significant insight into the regulation and mechanism of faithful DNA replication. Vaccinia's role as the agent used so successfully in the worldwide campaign to eradicate smallpox by vaccination, and its current potential as a recombinant vaccine engineered to carry foreign antigens, augments the importance of understanding the intricacies of viral replication.